Vitamin D Deficiencies

ABSTRACT

Methods for determining the amount of vitamin D compounds in a sample are provided. The methods can employ LC-MS/MS techniques and optionally the use of deuterated internal standards. Methods for diagnosing vitamin D deficiencies are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/709,780, filed Sep. 20, 2017 (now U.S. Pat. No. 10,416,149), which isa divisional of U.S. application Ser. No. 14/622,512 (now U.S. Pat. No.9,778,246), filed Feb. 13, 2015, which is a divisional of U.S.application Ser. No. 13/887,031 (now U.S. Pat. No. 8,987,002), filed May3, 2013, which is a continuation of U.S. application Ser. No. 13/656,005(now U.S. Pat. No. 8,592,218), filed Oct. 19, 2012, which is acontinuation application of U.S. application Ser. No. 13/021,825 (nowU.S. Pat. No. 8,349,613), filed Feb. 7, 2011, which is a continuation ofU.S. application Ser. No. 12/732,761 (now U.S. Pat. No. 7,901,944),filed on Mar. 26, 2010, which is a continuation of U.S. application Ser.No. 10/977,121 (now U.S. Pat. No. 7,700,365), filed on Oct. 29, 2004,all of which are incorporated by reference in their entirety.

TECHNICAL FIELD

This document relates to methods and materials for detecting vitamin Dcompounds (e.g., 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) in asample.

BACKGROUND

Vitamin D compounds are derived from dietary ergocalciferol (fromplants, vitamin D2) or cholecalciferol (from animals, vitamin D3), or byconversion of 7-dihydrocholesterol to vitamin D3 in the skin uponUV-exposure. Vitamin D2 and D3 are subsequently 25-hydroxylated in theliver to form 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3(25OHD3). 25OHD2 and 25OHD3 represent the main body reservoir andtransport form of vitamin D; they are stored in adipose tissue or aretightly bound by a transport protein while in circulation.

The exact levels of 25OHD2 and 25OHD3 that reflect optimal body storesare uncertain. Mild to modest deficiency can be associated withosteoporosis or secondary hyperparathyroidism. Severe deficiency maylead to failure to mineralize newly formed osteoid in bone, resulting inrickets in children and osteomalacia in adults.

Current immunoassay-based analytical methods for detecting 25OHD2 and25OHD3 cannot selectively differentiate between 25OHD2 and 25OHD3 andcan under-detect the amount of 25-OHD2. HPLC methods can use laborintensive extraction processes followed by long chromatographic runtimes.

SUMMARY

This document provides materials and methods that can be used to measure25OHD2 and 25OHD3 levels in a sample. For example, 25OHD2 and 25OHD3 canbe selectively detected and quantitated using mass spectrometrictechniques. The materials and methods are useful to aid in the diagnosisof vitamin D deficiencies or hypervitaminosis D, and to monitor vitaminD replacement therapies. In one embodiment, this document provides aLC-MS/MS method employing on-line sample extraction to allow for thesensitive, accurate, and precise quantification of 25OHD2, 25OHD3, orboth, in samples such as serum and plasma. Unlike manual immunoassays,the methods provided herein can be highly automated, can separatelymeasure 25OHD2 and 25OHD3, and can use an internal standard to monitorrecovery of the sample extraction process. In addition, the methods canprovide superior analytical performance as compared to immunoassays.

In general, one embodiment provides a method for determining an amountof 25-hydroxyvitamin D2 in a sample. The method includes using a massspectrometry technique to determine the amount of 25-hydroxyvitamin D2.The MS technique can employ atmospheric pressure chemical ionization.The mass spectrometry technique can be a tandem mass spectrometry(MS/MS) technique, or a LC-MS/MS technique. The LC can include anon-line extraction of the sample. The LC-MS/MS technique can include theuse of a triple quadrupole instrument in Multiple Reaction Monitoring(MRM), positive-ion mode, and can include a Q1 scan tuned to select aprecursor ion that corresponds to the [M+H⁺] of 25-hydroxyvitamin D₂.

In another embodiment, the amount of 25-hydroxyvitamin D3 can also bedetermined in addition to the amount of 25-hydroxyvitamin D2. In thisembodiment, an LC-MS/MS technique can include a Q1 scan tuned to select,independently, precursor ions that correspond to the [M+H⁺] of25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. An LC-MS/MS technique caninclude monitoring MRM precursor-product ion pair transitions having m/zvalues of 401.4/383.3 for 25-hydroxyvitamin D₃ and 413.0/395.3 for25-hydroxyvitamin D₂.

An internal standard, such as a deuterated 25-hydroxyvitamin D2 or D3,can be employed in any of the methods described herein. In certaincases, the internal standard is d₆-25-hydroxyvitamin D₃ having a MRMparent-daughter ion pair transition m/z value of 407.4/389.5.

A sample can be a biological or non-biological sample. A sample can be ahuman biological sample, such as a blood, urine, lachrymal, plasma,serum, or saliva sample.

In another embodiment, a method for determining whether or not a mammalhas a vitamin D deficiency is provided. The method includes determiningthe amount of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in a samplefrom the mammal. Any of the methods described herein can be used todetermine these amounts. A total amount of 25-hydroxyvitamin D2 and25-hydroxyvitamin D3 of <25 ng/mL indicates that the mammal has thevitamin D deficiency.

In yet another embodiment, a method for determining whether or not amammal has hypervitaminosis D is provided. The method includesdetermining the amount of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3in a sample from the mammal, where a total amount of 25-hydroxyvitaminD2 and 25-hydroxyvitamin D3 of >80 ng/mL indicates the mammal hashypervitaminosis D.

A method for monitoring vitamin D replacement therapy in a mammal isalso provided. The method includes determining the amount of25-hydroxyvitamin D2 in a sample from the mammal using one of themethods described herein. A lower concentration of 25-hydroxyvitamin D₂relative to the vitamin D replacement therapy is indicative of one ormore of the following: non-compliance with the replacement therapy,malabsorption of vitamin D supplements, and resistance to25-hydroxyvitamin D2.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims. Unless otherwisedefined, all technical and scientific terms used herein have the meaningcommonly understood by one of ordinary skill in the art to which thisinvention pertains. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. The disclosed materials, methods, andexamples are illustrative only and not intended to be limiting. Skilledartisans will appreciate that methods and materials similar orequivalent to those described herein can be used to practice theinvention.

DESCRIPTION OF DRAWINGS

FIG. 1 is a Q1 scan of 25OHD2 and 25OHD3.

FIG. 2A is a 25OHD2 product ion scan.

FIG. 2B is a 25OHD3 product ion scan.

DETAILED DESCRIPTION

Materials and methods for determining the amount of 25OHD2 and/or 25OHD3in a sample, such as a sample from a patient in a clinical setting, areprovided. The methods can be highly automated to allow for the efficientanalysis of a number of samples in minimal time. In addition, themethods can be highly sensitive and can allow for the accuratedifferentiation of 25OHD2 and 25OHD3, thus avoiding the under-detectionof one or both of the analytes by other methods. On-line extractionmethods can be employed, further minimizing sample handling andoptimizing run time.

A method described herein can include the use of mass spectrometrytechniques, such as tandem mass spectrometry (MS/MS) techniques. Incertain cases, a liquid chromatography tandem mass spectrometry(LS-MS/MS) technique can be used. A mass spectrometry technique caninclude the use of a triple quadrupole instrument in Multiple ReactionMonitoring, positive ion mode. Depending on the analyte of interest, aMS/MS technique can include a Q1 scan that is tuned to select precursorions that correspond to the positive ions ([M+H+]) of 25OHD2 and/or25OHD3. Precursor-product ion pairs transitions characteristic for25OHD2 and/or 25OHD3 can be monitored. An internal standard, such asdeuterated 25OHD3, can be added to any sample, e.g., to evaluate samplerecovery, precision, and/or accuracy.

Samples and Sample Preparation

A sample for analysis can be any sample, including biological andnon-biological samples. For example, a sample can be a food (e.g., meat,dairy, or vegetative sample) or beverage sample (e.g., orange juice ormilk). A sample can be a nutritional or dietary supplement sample. Incertain cases, a sample can be a biological sample, such as a tissue(e.g., adipose, liver, kidney, heart, muscle, bone, or skin tissue) orbiological fluid (e.g., blood, serum, plasma, urine, lachrymal fluid, orsaliva) sample. The biological sample can be from a mammal. A mammal canbe a human, dog, cat, primate, rodent, pig, sheep, cow, or horse.

A sample can be treated to remove components that could interfere withthe mass spectrometry technique. A variety of techniques known to thosehaving skill in the art can be used based on the sample type. Solidand/or tissue samples can be ground and extracted to free the analytesof interest from interfering components. In such cases, a sample can becentrifuged, filtered, and/or subjected to chromatographic techniques toremove interfering components (e.g., cells or tissue fragments). In yetother cases, reagents known to precipitate or bind the interferingcomponents can be added. For example, whole blood samples can be treatedusing conventional clotting techniques to remove red and white bloodcells and platelets. A sample can be de-proteinized. For example, aplasma sample can have serum proteins precipitated using conventionalreagents such as acetonitrile, KOH, NaOH, or others known to thosehaving ordinary skill in the art, optionally followed by centrifugationof the sample.

In certain cases, an internal standard can be added to a sample prior tosample preparation. Internal standards can be useful to monitorextraction/purification efficiency. For example, 25OHD2 and 25OHD3 canbind to serum proteins such as vitamin D-binding globulin. An internalstandard can be added to a sample and allowed to equilibrate for aperiod of time, e.g., 5, 10, 15, 20, 25, 30, 60, 120 or more minutes.Equilibration temperature can be from about 10° C. to about 45° C., orany value in between (e.g., 15, 25, 30, 35, 37, 42, or 44° C.). Incertain cases, equilibration can be at room temperature for about 15minutes.

An internal standard can be any compound that would be expected tobehave under the sample preparation conditions in a manner similar tothat of one or more of the analytes of interest. For example, astable-isotope-labeled version of an analyte of interest can be used,such as a deuterated version of an analyte of interest. While not beingbound by any theory, the physicochemical behavior of suchstable-isotope-labeled compounds with respect to sample preparation andsignal generation would be expected to be identical to that of theunlabeled analyte, but clearly differentiable by mass on the massspectrometer. In certain methods, deuterated 25OHD2 or deuterated 25OHD3can be employed. For example, d₆-25OHD3 can be used.

To improve run time and minimize hands-on sample preparation, on-lineextraction and/or analytical chromatography of a sample can be used.On-line extraction and/or analytical chromatography can be used, e.g.,in LC-MS/MS techniques. For example, in certain methods, a sample, suchas a deproteinized plasma sample, can be extracted using an extractioncolumn, followed by elution onto an analytical chromatography column.The columns can be useful to remove interfering components as well asreagents used in earlier sample preparation steps (e.g., to removereagents such as acetonitrile). Systems can be co-ordinated to allow theextraction column to be running while an analytical column is beingflushed and/or equilibrated with solvent mobile phase, and vice-versa,thus improving efficiency and run-time. A variety of extraction andanalytical columns with appropriate solvent mobile phases and gradientscan be chosen by those having ordinary skill in the art. Analytes thatelute from an analytical chromatography column can be then measured bymass spectrometry techniques, such as tandem mass spectrometrytechniques.

Mass Spectrometry

After sample preparation, a sample can be subjected to a massspectrometry (MS) technique. A mass spectrometry technique can useatmospheric pressure chemical ionization (APCI) in the positive ion modeto generate precursor positive ions. In APCI techniques, analytes ofinterest exist as charged species, such as protonated molecular ions[M+H⁺] in the mobile phase. During the ionization phase, the molecularions are desorbed into the gas phase at atmospheric pressure and thenfocused into the mass spectrometer for analysis and detection.Additional information relating to atmospheric pressure chemicalionization is known to those of skill in the art; see U.S. Pat. No.6,692,971.

MS analysis can be conducted with a single mass analyzer (MS) or a“tandem in space” analyzer such as a triple quadrupole tandem massspectrometer (MS/MS). Using MS/MS, the first mass filter (Quadrople 1,Q1) can select, or can be tuned to select, independently, one or more ofthe molecular ions of 25OHD2, 25OHD3, and the internal standard. Thesecond mass filter (Q3) is tuned to select specific product or fragmentions related to the analyte of interest. Between these two massfiltration steps, the precursor molecular ions can undergocollisionally-induced dissociation (CID) at Q2 to produce product orfragment ions. The previously-described mass spectrometry technique canalso be referred to as multiple reaction monitoring, or MRM. In multiplereaction monitoring, both quadrupoles Q1 and Q3 can be fixed (or tuned)each at a single mass, whereas Q2 can serve as a collision cell.

The precursor [M+H⁺] ions of 25OHD2 and 25OHD3 typically produce productions that reflect the loss of water from the sample. Accordingly,precursor-product ion pair transitions for 25OHD2 can have m/z values of413.0 and 395.3, while 25OHD3 can have precursor-product ion pairtransitions having m/z values of 401.4 and 383.3. Similarly, theinternal standard d6-25OHD3 can have precursor-product ion pairtransitions having m/z values of 407.4 and 389.5. The amount of each canbe determined by comparing the area of precursor or product transitions,or both, of 25OHD2 and/or 25OHD3, with those of a standard calibrationcurve, e.g., a standard calibration curve generated from a series ofdefined concentrations of pure 25OHD2 and/or 25OHD3 standards. Variablesdue to the extraction and the LC-MS/MS instrumentation can be normalizedby normalizing peak areas of the analyte of interest to the peak areasof the internal standard.

Any tandem MS machine and LC-MS/MS machine can be used, including theAPI 4000 triple quadrupole tandem mass spectrometer (ABI-SCIEX, Toronto,Canada). Software for tuning, selecting, and optimizing ion pairs isalso available, e.g., Analyst Software Ver. 1.4 (ABI-SCIEX).

Methods for Diagnosis

The methods described herein can be used in various diagnosticapplications to monitor vitamin D-related pathologies, vitamin D andcalcium homeostasis, and vitamin D replacement therapies. For example,the total amount of 25OHD2 and 25OHD3 in a sample, such as a humanpatient sample, can be compared with clinical reference values todiagnose a vitamin D deficiency or hypervitaminosis D. One set ofclinical reference values is set forth below, and represents clinicaldecision values that can apply to human males and females of all ages,rather than population-based reference values. Such population-basedreference values have been found to vary widely depending on ethnicbackground, age, geographic location of the studied population, andsampling season, and correlate poorly with concentrations associatedwith biologically and clinically relevant vitamin D effects.

Clinical reference values, total 25OHD2 and 25OHD3 <10 ng/mL Severedeficiency* 10 ng/mL-25 ng/mL Mild to moderate deficiency** 25 ng/mL-80ng/mL Optimum levels*** >80 ng/mL Toxicity possible *Could be associatedwith ostemalacia or rickets **May be associated with increased risk ofosteoporosis or secondary hyperparathyroidism ***Optimum levels in thenormal population **** 80 ng/mL is the lowest reported level associatedwith toxicity in patients without primary hyperparathyroidism who havenormal renal function. Most patients with toxicity have levels in excessof 150 ng/mL. Patients with renal failure can have very high 25OHD2 and25OHD3 levels without any signs of toxicity, as renal conversion to theactive hormones is impaired or absent.

In one embodiment, a method for determining whether or not a mammal hasa vitamin D deficiency is provided. The method can involve determiningthe amount of 25OHD2 and 25OHD3 in a sample from the mammal, such as ahuman. The amounts can be determined using any of the methods providedherein. Based on the clinical reference values set forth herein, a totalamount of 25OHD2 and 25OHD3 of less than 25 ng/mL in the sample canindicate that the mammal has a vitamin D deficiency.

In another embodiment, a method for determining whether or not a mammalhas hypervitaminosis D is provided. The method can involve determiningthe amount of 25OHD2 and 25OHD3 in a sample from the mammal. Based onthe clinical reference values set forth herein, a total amount of 25OHD2and 25OHD3 of more than 80 ng/mL in the sample can indicate that themammal has hypervitaminosis D.

In yet another embodiment, a method for monitoring vitamin D replacementtherapy in a patient is provided. The method can involve determining theamount of 25OHD2 in a sample from the patient using any of the methodsdescribed herein. A lower than expected concentration of 25OHD2 relativeto the amount expected from the replacement therapy can be indicative ofpatient non-compliance, malabsorption of vitamin D supplements, orresistance to 25OHD2.

EXAMPLES Example 1—Development of a High-Throughput LC-MS/MS Assay UsingOn-Line Extraction for the Measurement of 25OHD2 and 25OHD3

Materials

25OHD2 and 25OHD3 were purchased from Sigma (St. Louis, Mo.). Eachcompound was reconstituted separately in ethanol and analyzed forconcentration by UV Spectrophotometry at 264 nm using an ethanol blank.The analytes were combined in a stock solution and stored at −20° C.Working standards were prepared by diluting the stock solution instripped serum (Sera Care Inc.) with concentrations of 0-200 ng/mL(0-500 nmol/L).

Deuterated d₆-25OHD3 was purchased from AS VITAS (Norway) for use as aninternal standard. The compound was reconstituted in ethanol and storedat −20° C. A working internal standard solution was created by dilutingthe stock internal standard in 70% methanol containing 1 μg/mL estriol.

Sample Preparation

25 μL of working internal standard was added to 200 μL sample. Thesample was then incubated for 15 minutes at room temperature to allowthe internal standard time to equilibrate with any binding proteins inthe sample. Proteins were then precipitated by addition of 200 μL ofacetonitrile and separated from the supernatant by centrifugation. Thesupernatant was then transferred to a 96-deep-well plate and coveredwith a template film until analysis.

On-Line Extraction and LC-MS/MS

On-line extraction and HPLC chromatography of the supernatants wasperformed using a Cohesive Technologies TX4 Turbo Flow system with1.0×50 mm Cyclone extraction columns and 3.3 cm×4.6 mm, 3 μm LC-18(Supelco) analytical columns.

50 μL of the supernatant was injected onto the Cyclone extraction columnwith a mobile phase of 50% methanol, 0.005% formic acid at 4.0 mL/minfor 30 seconds. While the supernatant was injected onto the extractioncolumn, the LC-18 analytical column was equilibrated with 17.5%methanol, 0.005% formic acid at 0.75 mL/min. The analytes were theneluted from the extraction column for 90 seconds with methanol, 0.005%formic acid at 0.2 mL/min, mixed at a T-valve with 17.5% methanol,0.005% formic acid flowing at 0.55 mL/min to give a mobile phase of39.5% methanol, 0.005% formic acid, onto the analytical column. Therewas a step gradient to 87% methanol, 0.005% formic acid for theanalytical column, and the analytes were measured by tandem massspectrometry. The extraction column and analytical column were thenequilibrated with original conditions for 1 minute.

Analytes were analyzed on an API 4000 triple-quadrupole tandem massspectrometer (ABI-SCIEX, Toronto, Canada) using Analyst Software, Ver.1.4 (ABI-SCIEX). An atmospheric pressure chemical ionization (APCI)source was used at a temperature of 400° C. The MS/MS parameters werecurtain gas 10, GS1 22, CAD 6, NC 3, DP, 31, EP, 10, CE 13 and CXP 10.The ion transitions monitored were m/z 401.4/383.3 for 25OHD3, m/z413.0/395.3 for 25OHD2 and m/z 407.4/389.5 for d₆-25OHD3.

Samples

Serum separator (SST), serum-clot, EDTA, and sodium heparin tubes from 5normal volunteers were collected to assess stability and specimen type.Due to the lack of 25-OH D2 in normal donors samples, specimen-typesuitability was established by a recovery study on each sample type.Stability samples were also spiked prior to storage.

Linearity was established by diluting 5 elevated samples from theDiasorin RadioImmunoAssay (MA) (Stillwater, Minn.) in stripped serum(stripped of endogenous vitamin D). The 25OHD2 and 25OHD3 valuesobtained from the undiluted sample were used to calculate the expectedvalues of the diluted sample. The percentage of observed/expected wasthen calculated for each dilution and used as a measure of linearity.

To create quality control pools and establish precision, stripped serumwas separated into 3 pools and spiked with 25OHD2 and 25OHD3 to a low,medium and high level. Each pool was separated into 20-mL aliquots andfrozen at −80° C. One 20-mL aliquot was thawed, separated into 1.0-mLaliquots and refrozen at −20° C. Inter-assay precision was establishedby running an aliquot of each pool each day for 15 days. Sensitivity wasestablished by inter-assay precision on a low pool diluted in strippedserum. Intra-assay precision was established by running 20 separatesamples on each of 3 levels on the same assay.

A method comparison was done on 100 samples for the LC-MS/MS method, theDiasorin RIA, the ADVANTAGE automated chemiluminescent immunoassay(Nichols Diagnostics), and the LIASON automated chemiluminescentimmunoassay (Diasorin, Stillwater, Minn.). The samples were separatedinto 5 aliquots and frozen at −20° C. An aliquot was used for eachmethod.

LC-MS/MS Characteristics of 25OHD2 and 25OHD3

The Q1 scan (FIG. 1) and the Q3 scan (FIG. 2) of 25OHD2 (MW 412) and25OHD3 (MW 400) were obtained by infusing a 5 μg/mL solution at 10μL/min. The autotune mode of Analyst software was used to select andoptimize ion pairs. Ion pairs were also checked manually and found to bethe same. Each compound had an optimum Q1 ion corresponding to [M+H⁺]and an optimum Q3 ion corresponding to a H₂O loss. The deuteratedinternal standard d₆-25OHD3 gave a similar ion pair of m/z 407 to 389.

FIG. 3 shows a LC-MS/MS chromatogram of a sample. By collecting data fortwo minutes, the Cohesive system can collect data on one channel whileextracting another sample on the second channel.

Recovery data for each sample type is listed in Table 1. This datademonstrates that each specimen type is acceptable and that the strippedserum used for the standards is an acceptable standard matrix.

TABLE 1 Sensitivity, Low, Medium, High, n = 15 n = 15 n = 15 n = 1525OHD2 4.2 ng/mL 17 ng/mL 42 ng/mL 110 ng/mL CV 14.0% 5.0% 6.5% 5.7%25OHD3 1.7 ng/mL 24 ng/mL 55 ng/mL 132 ng/mL CV 12.9% 8.0% 7.4% 5.9%

The linearity data had a percent observed mean of 109% for 25OHD2 with arange of 93-125%. The 25OHD3 percent observed mean was 103% with a rangeof 96-118%. Sensitivity was also checked by linearity. One sample for25OHD2 diluted to 4.1 ng/mL had a percent observed of 96%, and onesample for 25OHD3 diluted to 1.3 ng/mL had a percent observed of 106%.

Precision data is provided in Table 1. 25OHD2 and 25OHD3 had inter-assayprecision of <10% in the low, medium and high range. Sensitivity wasestablished at 4 ng/mL for 25OHD2 and 2 ng/mL for 25OHD3 based on aninter-assay CV of <20% on the sensitivity pool.

25OHD2 and 25OHD3 were found to be stable at ambient and refrigeratedtemperatures for 7 days. Day 7 ambient samples (serum, EDTA plasma, andheparin plasma) had a mean difference of 7.6% from day 0 values with arange of −9.7% to 23.8%. Day 7 refrigerated samples (serum, EDTA plasmaand heparin plasma) had a mean difference of 1.6% from day 0 values witha range of −24.2% to 11.9%. Samples were also stable for 3 freeze/thawcycles. Samples (serum, EDTA plasma and heparin plasma) after 3freeze/thaw cycles had a mean difference of 0.6% from unfrozen freshvalues with a range of −11.5% to 11.5%.

Carry over was assessed by injecting stripped serum supernatants aftersupernatants of spiked samples (1720-8130 ng/mL) on each system. Threelevels of 25OHD2 and 25OHD3 were used with stripped serum run after eachlevel. The amount of detectable 25OHD2 and 25OHD3 in the stripped serumwas divided by the concentrations in the spiked samples. 25OHD2 had acarry over of 0.05%-0.14% and 25OHD3 had a carry over of 0.14%-0.24%.

Method comparison correlation data for the four assays tested is inTable 2. Slopes ranged from 1.01-1.03, and r values ranged from0.87-0.92 for samples that were 25OHD2 negative (only 25OHD3 present).Slopes ranged from 0.48-0.94 and r values ranged from 0.59-0.89 for25OHD2 positive patients (25OHD2 and 25OHD3 present), indicating theimmunoassays used in this comparison underestimate 25OHD2 levels.

TABLE 2 Assay Slope y-intercept r 25OHD2 Negative patients Diasorin RIA1.03 3.3 0.92 Liason 1.02 4.3 0.87 Advantage 1.01 3.3 0.89 25OHD2Positive patients Diasorin RIA 0.94 4.4 0.89 Liason 0.91 11.9 0.83Advantage 0.48 10.9 0.59

Linearity data demonstrate that the stripped serum is an acceptablematrix for standards. Values of the linearity study range from 211 ng/mLin an undiluted sample to as low as 1.3 ng/mL in a diluted sample. Thisdata reinforces the linear range of the standards 0-200 ng/mL.

Carry-over is negligible. Any sample following a sample >1000 ng/mL isrepeated to prevent any contamination.

The method comparison data shows good correlation for 25OHD3 samplesbetween all assays. The poor correlation of the Advantage assay in25OHD2 positive samples indicates the Advantage assay underestimates25OHD2.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for determining whether or not amammalian subject has a vitamin D deficiency, comprising a) obtaining abiological sample from the mammalian subject; b) deriving a sample fromthe biological sample by removing interfering components from thebiological sample, wherein the interfering components are removed fromthe biological sample by precipitation; c) determining an amount of25-hydroxyvitamin D2 simultaneously with an amount of 25-hydroxyvitaminD3 in a single sample derived from the mammalian subject's biologicalsample, wherein the determining comprises using a mass spectrometrytechnique to detect underivatized 25-hydroxyvitamin D2 and underivatized25-hydroxyvitamin D3 in the single sample, wherein the mass spectrometrytechnique comprises a LC-MS/MS technique and wherein the LC-MS/MStechnique comprise the use of a triple quadrupole instrument in MultipleReaction Monitoring (MRM), positive ion mode; d) comparing the amount oftotal 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the single samplewith clinical reference values indicative of vitamin D deficiency; ande) determining whether or not the mammalian subject has a vitamin Ddeficiency based on the comparisons in step d).
 2. The method of claim1, wherein the LC-MS/MS technique comprises a Q1 scan tuned to select aprecursor ion that corresponds to the [M+H+] of 25-hydroxyvitamin D2. 3.The method of claim 1, wherein the biological sample is a blood, urine,lachrymal, plasma, serum, or saliva sample.
 4. The method of claim 1,wherein the mammalian subject is a human.
 5. The method of claim 1,wherein the LC-MS/MS technique comprises atmospheric pressure chemicalionization.
 6. The method of claim 1, wherein the LC-MS/MS techniquecomprises a Q1 scan tuned to select, independently, precursor ions thatcorrespond to the [M+H+] of 25-hydroxyvitamin D2 and 25-hydroxyvitaminD3.
 7. The method of claim 1, wherein the LC-MS/MS technique comprisesmonitoring an MRM precursor-product ion pair transition having m/zvalues of 413.0/395.3 for 25-hydroxyvitamin D2.
 8. The method of claim1, wherein the LC-MS/MS technique comprises monitoringMRM-precursor-product ion pair transitions having m/z values of401.4/383.3 for 25-hydroxyvitamin D3 and 413.0/395.3 for25-hydroxyvitamin D2.
 9. The method of claim 1, further comprising usingan internal standard selected from the group consisting of a deuteratedderivative of 25-hydroxyvitamin D3 and a deuterated derivative of25-hydroxyvitamin D2.
 10. The method of claim 1, wherein the methodfurther comprise removing interfering components from the biologicalsample by one or more centrifugation, filtration, or chromatography. 11.The method of claim 10, wherein the chromatography is selected from thegroup consisting of on-line extraction chromatography, analyticalchromatography, and combinations thereof.
 12. The method of claim 1,wherein the method further comprises removing interfering componentsfrom the biological sample de-proteinizing the biological sample. 13.The method of claim 1, wherein the method comprises determining theamounts of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using astandard calibration curve.
 14. The method of claim 13, furthercomprising using an internal standard selected from the group consistingof a deuterated derivative of 25-hydroxyvitamin D3 and a deuteratedderivative of 25-hydroxyvitamin D2.
 15. The method of claim 1, whereinthe clinical reference value for a vitamin D deficiency is a totalamount of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 of less than 25ng/mL.